Spider peptide toxins as leads for drug development

Venomous animals use a highly complex cocktails of proteins, peptides and small molecules to subdue and kill their prey. As such, venoms represent highly valuable combinatorial peptide libraries, displaying an extensive range of pharmacological activities, honed by natural selection. Modern analytical technologies enable us to take full advantage of this vast pharmacological cornucopia in the hunt for novel drug leads. Spider venoms represent a resource of several million peptides, which selectively target specific subtypes of ion channels. Structure–function studies of spider toxins are leading not only to the discovery of novel molecules, but also to novel therapeutic routes for cardiovascular diseases, cancer, neuromuscular diseases, pain and to a variety of other pathological conditions. This review presents an overview of spider peptide toxins as candidates for therapeutics and focuses on their applications in the discovery of novel mechanisms of analgesia.     

Amino acid/spermine conjugates: polyamine amides as potent spermidine uptake inhibitors

In this paper we describe the synthesis and characterization of a series of simple spermine/amino acid conjugates, some of which potently inhibit the uptake of spermidine into MDA-MB-231 breast cancer cells. The presence of an amide in the functionalized polyamine appeared to add to the affinity for the polyamine transporter. The extensive biological characterization of an especially potent analogue from this series, the Lys-Spm conjugate (31), showed this molecule will be an extremely useful tool for use in polyamine research. It was shown that the use of 31 in combination with DFMO led to a cytostatic growth inhibition of a variety of cancer cells, even when used in the presence of an extracellular source of transportable spermidine. It was furthermore shown that this combination effectively reduced the cellular levels of putrescine and spermidine while not affecting the levels of spermine. These facts together with the nontoxic nature of 31 make it a novel lead for further anticancer development.     

GABA, glutamate and substance P-like immunoreactivity release: effects of novel GABAB antagonists.

1. The effects of various GABA receptor ligands on the electrically-evoked release of endogenous GABA, glutamate and substance P-like immunoreactivity from the dorsal horn of rat isolated spinal cord were examined. 2. Exogenous GABA (10-300 microM) significantly decreased the evoked, but not basal, release of endogenous glutamate in a concentration-dependent manner. The GABAA agonist, isoguvacine (1-100 microM), failed to decrease the release of glutamate although it did reduce the release of GABA. Baclofen (0.1-1000 microM), the GABAB agonist, reduced the release of GABA and glutamate in a stereospecific and concentration-dependent manner. 3. The actions of five GABAB antagonists on these release systems were compared. CGP36742, CGP52432, CGP55845A and CGP57250A significantly increased the evoked release of GABA and glutamate. They also reversed the effects of (-)-baclofen in a concentration-dependent manner. On the other hand, while CGP56999A had no effect on glutamate release, it was an effective antagonist of the baclofen-induced inhibition of GABA and substance P release. 4. These results suggest that GABAB receptors on nerve terminals within the dorsal horn spinal cord may be heterogeneous. However, this is based solely on the data obtained with CGP56999A which affected only GABA and substance P, but not glutamate, release.     

Therapeutic potential of novel glutamate receptor antagonists in migraine

Background: Migraine is a common and disabling neurological disorder. Although the pharmacotherapy of migraine has advanced in parallel with our understanding of the pathophysiology of the disease, there is still a considerable unmet need to find more effective treatments. Migraine pathophysiology involves activation or the perception of activation of the trigeminovascular system. Glutamate, the major excitatory neurotransmitter in the CNS, is implicated in elements of the pathophysiology of the disorder, including trigeminovascular activation, central sensitization and cortical spreading depression. Objective: The aim of this article is to review the potential use of glutamate receptor antagonists as innovative neuronally targeted treatments of migraine. Methods: A systematic search of peer-reviewed publications was performed in PubMed on glutamate and migraine/trigeminovascular activation, and important references providing an insight into migraine pathophysiology are included. The results of unpublished trials were obtained from presentations at national and international meetings. Results/conclusions: The preclinical and clinical data argue strongly for a role of glutamatergic receptor activation in migraine. The pharmacology of glutamatergic trigeminovascular responses in brain areas involved in migraine pathophysiology is relevant to the development of new therapies for this disabling condition. Glutamate receptors represent a promising target for a valuable, non-vasoconstrictor, and perhaps more importantly neuronal-specific therapeutic approach to the treatment of migraine.     

The alpha v integrin antagonists as novel anticancer agents: an update

Integrins are involved in many cellular processes, including some pathological ones associated with various cancers, both solid tumours and metastases. Since integrins are involved in such critical processes as gene expression, which lead to cellular proliferation, migration, survival and angiogenesis, they represent potential targets for therapeutic intervention. The alpha(v)beta(3) integrin is one of the most widely studied integrins because it is one of the most promiscuous. Published studies provide compelling evidence that small molecule antagonists have the potential to treat both solid tumours and metastases, serve as diagnostic imaging agents and be used for site-directed delivery of drugs to solid tumours. The alpha(v)beta(3) integrin antagonists also inhibit blood vessel formation associated with tumour growth. Therapeutic candidates have included antibodies, cyclic peptides, peptidomimetics and small molecules. A number of potent small-molecule antagonists of the alpha(v)beta(3) integrin have now been identified and are progressing in the clinic. This review focuses on the role of alpha(v)beta(3) in cancer. The rationale for the development of the therapeutic and diagnostic candidates based on the key role of alpha(v)beta(3) is discussed.     

6-arylcoumarins as novel nonsteroidal type progesterone antagonists: an example with receptor-binding-dependent fluorescence

Various 6-arylcoumarin derivatives were designed and synthesized as candidate nonsteroidal type progesterone antagonists. 6-Bromocoumarin derivatives were prepared from the corresponding 4-substituted 2-acetoxy-5-bromobenzaldehyde by employing the Still-Gennari modification of the Horner-Wadsworth-Emmons olefination reaction and were converted to 6-arylcoumarins by means of Suzuki-Miyaura cross-coupling reactions. The biological activities of these coumarin derivatives were evaluated by means of alkaline phosphatase assay in the T47D human breast carcinoma cell line. Among the synthesized compounds, 36 (IC(50) = 0.12 μM) and 38 (IC(50) = 0.065 μM), bearing a five-membered heterocycle, showed potent PR antagonist activity. Competitive binding assay showed that compounds 8 and 34 have potent PR binding affinity. The fluorescence of compound 8 was dependent on the solvent properties and was increased in the presence of PR ligand binding domain. This property might be applicable to the development of fluorescence probes for studies on PR.     

Muscarinic toxins: novel pharmacological tools for the muscarinic cholinergic system.

Muscarinic receptors are widely spread throughout the body, and are involved in the regulation of fundamental physiological processes, like the modulation of the heart rate, control of motor systems and modulation of learning and memory. In the central nervous system the cholinergic transmission is mainly mediated by muscarinic receptors; there are five subtypes that are all expressed in the brain of mammals (m1-m5). There are regional differences in their concentrations in the brain and more than one subtype is expressed in the same cell. It has been difficult to study their localization and function in vivo due to the lack of ligands that exclusively act on one subtype of the receptor. We studied the action of the muscarinic toxins MT1, MT2 and MT3, from the venom of the snake Dendroaspis angusticeps, on muscarinic receptors, by using the classical muscarinic radioligand 3H-NMS as reporter of the inhibition of its own binding, to either native or cloned receptors. We have also studied the in vivo effects on memory retention of the injection of the toxins into discrete brain regions. The muscarinic toxins appear to be invaluable tools to study receptor pharmacology, physiology and structure/function relationships. They would enable the design of new, more selective, pharmacological agents.     

alpha-Conotoxins: nicotinic acetylcholine receptor antagonists as pharmacological tools and potential drug leads

Conotoxins are small disulfide rich peptides from the venoms of marine cone snails. They target specific nicotinic acetylcholine receptor (nAChR) subtypes with high affinity and potency and are therefore valuable as neuropharmacological probes and potential drug leads. This article gives a general overview of the chemical and biological features of alpha-conotoxins, including their pharmacology, binding interactions and structure. A detailed analysis of recently reported three-dimensional structures from members of different subfamilies of the alpha-conotoxins, including those with 3/5, 4/3, 4/6 and 4/7 spacings of their two intracysteine loops is given. The structures are generally well defined and represent useful frameworks for the display of amino acid residues to target molecules.     

Informative library design as an efficient strategy to identify and optimize leads: application to cyclin-dependent kinase 2 antagonists

The application of an informative, iterative library design strategy is presented for lead identification and optimization. The computational algorithm underlying informative design systematically uses data from both active and inactive compounds and maximizes the information gained from subsequent design-synthesis-screening cycles. Retrospective analysis of a released dataset of 17 550 compounds and corresponding cyclin-dependent kinase-2 activities showed that informative library design yields significant enrichments of active compounds and efficiently discovers novel chemotypes in comparison with commonly used diversity-similarity protocols.     

Southern African scorpion toxins: an overview.

In southern Africa there are 130 species of scorpions and only a few species' venom have been investigated to date. This review gives an overview of the research done on the venom of southern African scorpions and the toxins and peptides identified up to date. It also aims to highlight the enormous potential that lies in this field of research. Southern African scorpion toxins include four long-chain Na(+)-channel toxins, four short-chain alpha-K(+)-channel toxins (alpha-KTx), three Ca(2+)-channel toxins and also an insect-selective peptide active on K(+) and Cl(-) channels. Three antimicrobial peptides have also been isolated and characterized. All of these peptides are diverse not only in function and target but also in the species they are isolated from.     

Metabotropic glutamate receptors as novel targets for anxiety and stress disorders

Anxiety and stress disorders are the most commonly occurring of all mental illnesses, and current treatments are less than satisfactory. So, the discovery of novel approaches to treat anxiety disorders remains an important area of neuroscience research. Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system, and G-protein-coupled metabotropic glutamate (mGlu) receptors function to regulate excitability via pre- and postsynaptic mechanisms. Various mGlu receptor subtypes, including group I (mGlu(1) and mGlu(5)), group II (mGlu(2) and mGlu(3)), and group III (mGlu(4), mGlu(7) and mGlu(8)) receptors, specifically modulate excitability within crucial brain structures involved in anxiety states. In addition, agonists for group II (mGlu(2/3)) receptors and antagonists for group I (in particular mGlu(5)) receptors have shown activity in animal and/or human conditions of fear, anxiety or stress. These studies indicate that metabotropic glutamate receptors are interesting new targets to treat anxiety disorders in humans.     

Cytoskeleton as an emerging target of anthrax toxins

Bacillus anthracis, the agent of anthrax, has gained virulence through its exotoxins produced by vegetative bacilli and is composed of three components forming lethal toxin (LT) and edema toxin (ET). So far, little is known about the effects of these toxins on the eukaryotic cytoskeleton. Here, we provide an overview on the general effects of toxin upon the cytoskeleton architecture. Thus, we shall discuss how anthrax toxins interact with their receptors and may disrupt the interface between extracellular matrix and the cytoskeleton. We then analyze what toxin molecular effects on cytoskeleton have been described, before discussing how the cytoskeleton may help the pathogen to corrupt general cell processes such as phagocytosis or vascular integrity.     

Studies on neurokinin antagonists. 1. The design of novel tripeptides possessing the glutaminyl-D-tryptophylphenylalanine sequence as substance P antagonists.

To discover a novel and low molecular weight substance P (SP) antagonist we postulated that the essential binding domain of peptide ligands was only a small portion in the whole structure. On the basis of this assumption, we selected the known octapeptide SP antagonist D-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Phe-NH2 (1) as a lead and synthesized its fragment tripeptides which were evaluated for their activity to block 3H-SP binding on guinea pig lung membranes. The protected tripeptide N alpha-[N alpha-[N alpha-(tert-butyloxycarbonyl)-L-glutaminyl]-N1-formyl-D-tryptophyl]- L-phenylalanine benzyl ester [Boc-Gln-D-Trp(CHO)-Phe-OBzl (4a)], corresponding to the Gln-D-Trp-Phe part of 1, exhibited 7-fold potent inhibitory activity in comparison with 1. Studies on structure-activity relationships revealed that the D-tryptophan, L-phenylalanine, and benzyl ester were quite important to maintain the high binding affinity. It was also indicated that 4a antagonized the SP-induced contraction of isolated guinea pig trachea strips (IC50 = 4.7 x 10(-6) M).     

Discovery of aryloxy tetramethylcyclobutanes as novel androgen receptor antagonists

An aryloxy tetramethylcyclobutane was identified as a novel template for androgen receptor (AR) antagonists via cell-based high-throughput screening. Follow-up to the initial "hit" established 5 as a viable lead. Further optimization to achieve full AR antagonism led to the discovery of 26 and 30, both of which demonstrated excellent in vivo tumor growth inhibition upon oral administration in a castration-resistant prostate cancer (CRPC) animal model.     

Angiotensin II-receptor antagonists: an overview.

Angiotensin II (AT-II)-receptor antagonists are reviewed. Research focused on blocking the renin-angiotensin system (RAS) led to the discovery of angiotensin-converting-enzyme (ACE) inhibitors, which are effective in the treatment of hypertension but are associated with a high frequency of cough and other adverse effects. AT-II-receptor antagonists were developed as agents that would more completely block the RAS and thus decrease the adverse effects seen with ACE inhibitors. AT-II-receptor antagonists include losartan, valsartan, irbesartan, candesartan, eprosartan, telmisartan, and tasosartan. Several clinical trials have demonstrated that AT-II-receptor antagonists are as effective as calcium-channel blockers, beta-blockers, and ACE inhibitors in the treatment of hypertension and induce fewer adverse effects. The adverse effects of AT-II-receptor antagonists--dizziness, headache, upper-respiratory-tract infection, cough, and gastrointestinal disturbances--occur at about the same rate as with placebo. [corrected]. All available AT-II-receptor antagonists seem to be equally effective in reducing both systolic and diastolic blood pressure, and they are comparable in cost. Currently, AT-II-receptor antagonists are used either as monotherapy in patients who cannot tolerate ACE inhibitors or in combination with other antihypertensive agents. Angiotensin II-receptor antagonists are well tolerated and are as effective as ACE inhibitors in decreasing blood pressure.     

Mosquito inoculation: an alternative bioassay for toxins

Mosquitoes were evaluated as a bioassay host for several classes of biological toxins. Mosquitoes were sensitive to snake toxic or neurotoxic phospholipase A2 enzymes (but not to nontoxic phospholipase A2 enzymes), cobrotoxin, saxitoxin, microcystin and the scorpion insect sodium channel toxin. Mosquitoes were not sensitive to ricin, diphtheria toxin, anthrax toxin, botulinum toxin, tetanus toxin, conotoxin G or a scorpion sodium channel toxin toxic to mammals. Specific antisera neutralization tests with mosquitoes gave comparable results to those of a mouse assay. The mosquito is a suitable bioassay animal for many, but not all biological toxins, and offers a safer, more efficient and economical assay than mice.     

Discovery of novel heteroarylazoles that are metabotropic glutamate subtype 5 receptor antagonists with anxiolytic activity

The highly potent, selective, and brain-penetrant metabotropic glutamate subtype 5 (mGlu5) receptor antagonists 3-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile (47) and 3-fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile (48) are reported. Compound 47 is active in the rat fear-potentiated startle (FPS) model of anxiety with ED(50) = 5.4 mg/kg (po) when dosed acutely. In this model the anxiolytic effects of 47 rapidly tolerate on repeated dosing.     

Reduction of desensitization of a glutamate ionotropic receptor by antagonists.

The glutamate receptor channel subtype that responds to both quisqualate (QA) and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) was expressed in Xenopus oocytes injected with rat cerebral cortex mRNA. Voltage-clamp current responses to QA, AMPA, and glutamate (GLU) exhibited a rapid increase followed by a decrease to a desensitized steady state (DS). Perfusion with high agonist concentrations produced smaller DS responses than perfusion with low concentrations. During the DS, the current was increased by lowering of the concentration of agonist or by application of low concentrations of a competitive antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX). This paradoxical increase of the agonist-induced currents during the DS was also observed in cultured Purkinje cells with another competitive antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). Dose-response curves obtained in oocytes were bell shaped, with a negative slope for high concentrations of QA. DNQX shifted these bell-shaped curves to the right. Together, these results indicate that the agonists are able to reversibly inhibit the AMPA receptor. The classical desensitization model of Katz and Thesleff [J. Physiol. (Lond.) 138:63-80 (1957)] cannot account for our observations.     

Berbanes: search for novel alpha-2 adrenoceptor antagonists

We have previously described the alpha-2 adrenoceptor antagonist properties of berbanes and its stereoisomers. Of the compounds studied CH-38083 (2,3-methylenedioxy-11-betahydroxyalloberbane) has been selected for further analysis based upon its high affinity and selectivity for central and peripheral alpha-2 adrenoceptors. Structure activity relationship study revealed that the aromatic ring with its substituents at C-2 and C-3 positions, the nitrogen atom, the hydroxy group at C-11 position and the methoxycarbonyl group at C-12 position are important for the binding of the berbanes to the alpha-2 adrenoceptors. Using alloberbane derivatives for characterization of the alpha-2 adrenoceptors it was speculated that xylazine sensitive alpha-2 adrenoceptors in the rat vas deferens and in the guinea-pig ileum are similar, whereas xylazine sensitive and noradrenaline sensitive alpha-2 adrenoceptors of the guinea-pig ileum may belong to different subtypes. Correlation studies indicated that modification of the molecular structure of the alloberbanes can lead to either increased or decreased alpha-2 adrenoceptor antagonists activity without parallel changes in the alpha-1 adrenoceptor antagonist potency. The low affinity of CH-38083 for other receptor populations (muscarinic, histamine, dopamine receptors) makes this compound attractive for investigation of alpha-2 adrenoceptor-mediated neural processes in the central nervous system and periphery.